Processing by OmpT of fusion proteins carrying the HlyA transport signal during secretion by the Escherichia coli hemolysin transport system.

Summary A fusion gene (ces-hlyA _s) was constructed by ligating the genetic information for the C-terminal 60 amino acids (hlyA _s) ofEscherichia coli hemolysin (H1yA) to the ces gene for a cholesterol esterase/lipase (CE) from aPseudomonas species. Part (about 30 %) of the expressed fusion protein CE-H1yA_s was secreted inE. coli carryinghlyB andhlyD genes. Following the insertion between the reporter gene andhlyA _s of a linker sequence that contains the information for potential cleavage sites for the outer membrane protease OmpT, two different fusion proteins (PhoA-H1yA_s and CE-HlyA_s) were shown to be cleaved by OmpT between the two parts during H1yB/H1yD-mediated secretion. Processed PhoA and CE accumulated in the supernatant. The efficiency of cleavage by OmpT was considerably improved by increasedompT gene dose. It was further shown that OmpT preferentially recognizes potential cleavage sites within the linker sequence.     

Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins

A simple and efficient procedure for the construction of secreted fusion proteins inEscherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) ofE. coli hemolysin (HlyA). This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini. For the construction of model gene fusions we usedlacZ, encoding the cytoplasmicβ-galactosidase (β-Gal), andphoA, encoding the periplasmic alkaline phosphatase, as target genes. Our data suggest that allβ-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions. In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in asecA mutant strain only under SecA-deficient conditions.     

Extracellular PagC-HlyAS fusion protein for the generation and identification of Salmonella-specific antibodies

The outer membrane protein, PagC, of Salmonella typhimurium was converted into a secreted protein by linking the 61-amino-acid long, C-terminal signal sequence of the E. coli hemolysin protein (HlyA_S) to the mature PagC peptide. This PagC-HlyA_S fusion protein was expressed and efficiently secreted into the culture supernatant by E. coli upon complementation with the hemolysin secretion proteins HlyB and HlyD. Polyclonal antibodies raised against this fusion protein not only recognized PagC in the membrane fraction of all salmonellae by Western blotting, but also reacted with proteins of smaller size in other gram-negative bacteria tested. A monoclonal antibody against the PagC-HlyA_S fusion protein recognized only PagC in membrane fractions. The antibody-binding domain was determined using synthetic peptides derived from specific PagC domains. Sera from Salmonella-infected human patients and from a rabbit infected with S. typhimurium did not react with PagC in immunoblots, suggesting that PagC may not be recognized as a major antigen by the humoral immune system. Received: 16 August 1995/Received revision: 6 November 1995/Accepted: 10 November 1995     

Correlation between the Presence of Virulence-Associated Genes as Determined by PCR and Actual Virulence to Mice in Various Strains of Listeria Spp.

Five chromosomal genes, prfA, plcA, hlyA, mpl and plcB, are implicated in the virulence of Listeria monocytogenes and some of these genes have been used for the identification of bacteria by polymerase chain reaction (PCR). Using 6 strains of L. monocytogenes and 3 L. innocua strains, the relationship was examined between the presence of five virulence-associated genes and actual virulence to mice in terms of 50% lethal dose (LD₅₀), bacterial viability in the organ of infected mice and the intracellular growth in cultured macrophages. None of the five genes could be amplified by PCR in all the L. innocua strains and they were actually avirulent to mice. All L. monocytogenes strains were shown to be virulent and to have intact virulence-associated genes except for the strain ATCC15313. This particular strain was revealed to be avirulent and defective in hlyA and plcA in PCR amplification. It was suggested that PCR detection of genes prfA, mpl, or plcB may not be sufficient to detect virulent strains of L. monocytogenes. It appeared that the ability to produce listeriolysin O (LLO), which is encoded by hlyA, was critical for the expression of virulence regardless of the amount of LLO produced.     

Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins

A simple and efficient procedure for the construction of secreted fusion proteins inEscherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) ofE. coli hemolysin (HlyA). This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini. For the construction of model gene fusions we usedlacZ, encoding the cytoplasmic-galactosidase (-Gal), andphoA, encoding the periplasmic alkaline phosphatase, as target genes. Our data suggest that all-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions. In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in asecA mutant strain only under SecA-deficient conditions.     

In Vitro Comparison of the Effects of Probiotic,Commensal and Pathogenic Strains on Macrophage Polarization

Macrophages are important with respect to both innate and adaptive immune responses and are known to differentiate into pro-inflammatory M1- or anti-inflammatory M2-phenotypes following activation. In order to study how different bacteria affect macrophage polarization, we exposed murine RAW 264.7 macrophages to sixteen different strains representing probiotic strains, pathogens, commensals and strains of food origin. Increased inducible nitric oxide synthase (iNOS) or arginase-1 gene expression indicates M1 or M2 polarization, respectively, and was quantified by qRT-PCR. Strains of Escherichia and Salmonella elevated iNOS expression more so than strains of Enterococcus, Lactobacillus and Lactococcus, indicating that Gram-negative strains are more potent M1 inducers. However, strain-specific responses were observed. For instance, Escherichia coli Nissle 1917 was a poor inducer of iNOS gene expression compared to the other E. coli strains, while Enterococcus faecalis Symbioflor-1 was more potent in this respect compared to all the eleven Gram-positive strains tested. Macrophage polarization was further characterized by quantifying secreted pro- and anti-inflammatory cytokines. Exposure to the pathogen E. coli 042 produced a cytokine profile indicating M1 differentiation, which is in accordance with the PCR data. However, exposure to most strains resulted in either high or low secretion levels of all cytokines tested, rather than a clear M1 or M2 profile. In general, the Gram-negative strains induced high levels of cytokine secretion compared to the Gram-positive strains. Interestingly, strains of human origin had a higher impact on macrophages compared to strains of food origin.     

Multiplex PCR and DNA array for the detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. targeting virulence-related genes

A multiplex PCR and DNA array for quick detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. was developed using specific genetic markers derived from virulence-related genes. The genetic markers of cytK, sei, prfA, rfB, and hilA gene specifically amplified DNA fragments of 320 bp, 500 bp, 700 bp, 1.0 kb and 1.2 kb from B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., respectively. These markers are specific for the detection of the corresponding target pathogens. The sensitivity of the genetic markers was down to ～0.5 fg genomic DNA and ～10¹ CFU/ml (one bacterial cell per reaction) of bacterial culture. The combination of mPCR and DNA macroarray hybridization sensitively and specifically detected B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., in complex mixed cultures and food matrices. Thus, this mPCR and macroarray-based approach serves as rapid and reliable diagnostic tool for the detection of these five pathogens.     

Change in the cellular localization of alkaline phosphatase by alteration of its carboxy-terminal sequence

Summary Alkaline phosphatase (AP) is secreted into the medium when the carboxy-terminal 25 amino acids are replaced by the 60 amino acid carboxy-terminal signal peptide (HlyA_s) ofEscherichia coli haemolysin (HlyA). Secretion of the AP-HlyA_s fusion protein is dependent on HlyB and HlyD but independent of SecA and SecY. The efficiency of secretion by HlyB/HlyD is decreased when AP carries its own N-terminal signal peptide. Translocation of this fusion protein into the periplasm is not observed even in the absence of HlyB/HlyD. The failure of the Sec export machinery to transport the latter protein into the periplasm seems to be due in part to the loss of the carboxy-terminal sequence of AP since even AP derivatives which do not carry the HlyA signal peptide but lack the 25 C-terminal amino acids of AP are localized in the membrane but not translocated into the periplasm.     

Improved Secretory Production of Recombinant Proteins by Random Mutagenesis of hlyB, an Alpha-Hemolysin Transporter from Escherichia coli

Fusion proteins with an alpha-hemolysin (HlyA) C-terminal signal sequence are known to be secreted by the HlyB-HlyD-TolC translocator in Escherichia coli. We aimed to establish an efficient Hly secretory expression system by random mutagenesis of hlyB and hlyD. The fusion protein of subtilisin E and the HlyA signal sequence (HlyA₂₁₈) was used as a marker protein for evaluating secretion efficiency. Through screening of more than 1.5 × 10⁴ E. coli JM109 transformants, whose hlyB and hlyD genes had been mutagenized by error-prone PCR, we succeeded in isolating two mutants that had 27- and 15-fold-higher levels of subtilisin E secretion activity than the wild type did at 23°C. These mutants also exhibited increased activity levels for secretion of a single-chain antibody-HlyA₂₁₈ fusion protein at 23 and 30°C but unexpectedly not at 37°C, suggesting that this improvement seems to be dependent on low temperature. One mutant (AE104) was found to have seven point mutations in both HlyB and HlyD, and an L448F substitution in HlyB was responsible for the improved secretion activity. Another mutant (AE129) underwent a single amino acid substitution (G654S) in HlyB. Secretion of c-Myc-HlyA₂₁₈ was detected only in the L448F mutant (AE104F) at 23°C, whereas no secretion was observed in the wild type at any temperature. Furthermore, for the PTEN-HlyA₂₁₈ fusion protein, AE104F showed a 10-fold-higher level of secretion activity than the wild type did at 37°C. This result indicates that the improved secretion activity of AE104F is not always dependent on low temperature.     

A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157:H7

A multiplex PCR procedure that detects six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in Escherichia coli O157:H7 was developed. Analyses of the available sequences of the six major virulence genes and the published primers allowed us to develop the six-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting six bands for fliC, stx1, stx2, eae, rfbE, and hlyA were even and distinct with product sizes of 949, 655, 477, 375, 296, and 199 bp, respectively. The procedure was validated with a total of 221 E. coli strains that included 4 ATCC, 84 cattle, and 57 human E. coli O157:H7 strains as well as 76 non-O157 cattle and human E. coli strains. The results of all 221 strains were similar to the results generated by established multiplex PCR methods that involved two separate reactions to detect five virulence genes (stx1, stx2, eae, fliC, and hlyA). Specificity of the O antigen was indicated by amplification of only O157, and not O25, O26, O55, O78, O103, O111, O127, and O145 E. coli serotypes. Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 10⁴ CFU/g (10 cells/reaction) of E. coli O157. After a 6-h enrichment of E. coli O157-spiked samples, a sensitivity level of 10 CFU/g was achieved.     

Fluorescent reference strains of bacteria by chromosomal integration of a modified green fluorescent protein gene

Fluorescent reference strains of bacteria carrying a stable chromosomally integrated single copy of the gfp gene have been developed. A modified version of the gfp gene has been generated by mutagenesis and expressed under the control of the bacteriophage lambda promoter P_L. A cassette comprising bacteriophage Mu transposon arms flanking the modified gfp gene and regulatory regions was irreversibly integrated as an in-vitro-assembled transposition complex into the genomes of Escherichia coli and Salmonella spp. The modified green fluorescent protein (GFP) protein retained the fluorescence excitation and emission wavelengths of wild-type GFP. However, it fluoresced more brightly in E. coli and Salmonella compared to wild-type GFP, presumably due to improved protein maturation. Fluorescent E. coli and Salmonella strains carrying the gfp gene cassette were easily differentiated from their respective non-fluorescent parental strains on various growth media by visualization under UV light. The bacterial strains produced by this method remained viable and stably fluorescent when incorporated into a matrix for delivery of exact numbers of viable bacterial cells for use as quality control agents in microbiological procedures.     

Detection of the Emerging Shiga Toxin-Producing Escherichia coli O26:H11/H? Sequence Type 29 (ST29) Clone in Human Patients and Healthy Cattle in Switzerland

Shiga toxin-producing Escherichia coli O26:H11/H⁻ strains showing the characteristics of the emerging human-pathogenic ST29 clone (stx_2a⁺ only, eae⁺, plasmid gene profile hlyA⁺ etpD⁺) were detected from human patients and healthy cattle, indicating a possible reservoir. Sheep also appear to shed strains related to the new pathogenic clone O26:H11/H⁻ (ST29, stx_1a⁺ only, eae⁺, plasmid gene profile hlyA⁺ etpD⁺).     

Oral Delivery of a Novel Attenuated Salmonella Vaccine Expressing Influenza A Virus Proteins Protects Mice against H5N1 and H1N1 Viral Infection

Attenuated strains of invasive enteric bacteria, such as Salmonella, represent promising gene delivery agents for nucleic acid-based vaccines as they can be administrated orally. In this study, we constructed a novel attenuated strain of Salmonella for the delivery and expression of the hemagglutinin (HA) and neuraminidase (NA) of a highly pathogenic H5N1 influenza virus. We showed that the constructed Salmonella strain exhibited efficient gene transfer activity for HA and NA expression and little cytotoxicity and pathogenicity in mice. Using BALB/c mice as the model, we evaluated the immune responses and protection induced by the constructed Salmonella-based vaccine. Our study showed that the Salmonella-based vaccine induced significant production of anti-HA serum IgG and mucosal IgA, and of anti-HA interferon-γ producing T cells in orally vaccinated mice. Furthermore, mice orally vaccinated with the Salmonella vaccine expressing viral HA and NA proteins were completely protected from lethal challenge of highly pathogenic H5N1 as well as H1N1 influenza viruses while none of the animals treated with the Salmonella vaccine carrying the empty expression vector with no viral antigen expression was protected. These results suggest that the Salmonella-based vaccine elicits strong antigen-specific humoral and cellular immune responses and provides effective immune protection against multiple strains of influenza viruses. Furthermore, our study demonstrates the feasibility of developing novel attenuated Salmonella strains as new oral vaccine vectors against influenza viruses.     

Cytotoxicity Potential and Genotypic Characterization of Escherichia coli Isolates from Environmental and Food Sources

The presence of Escherichia coli isolates in the environment is a potential source of contamination of food and water supplies. Moreover, these isolates may harbor virulence genes that can be a source of new forms of pathogenic strains. Here, using multiplex PCR, we examined the presence of virulence gene markers (stx₁, stx₂, eaeA, hlyA) in 1,698 environmental isolates of E. coli and 81 isolates from food and clinical sources. The PCR analysis showed that ~5% (79 of 1,698) of the total environmental isolates and 96% (79 of 81) of the food and clinical isolates were positive for at least one of the genes. Of the food and clinical isolates, 84% (68 of 81 isolates) were positive for all four genes. Of the subset of environmental isolates chosen for further analysis, 16% (13 of 79 isolates) were positive for stx₂ and 84% (66 of 79 isolates) were positive for eaeA; 16 of the latter strains were also positive for hlyA. The pathogenic potentials of 174 isolates (81 isolates from food and clinical sources and 93 isolates from environmental sources) were tested by using a cytotoxicity assay based on lactate dehydrogenase release from Vero cells. In general, 97% (79 of 81) of the food and clinical isolates and 41% (39 of 93) of the environmental isolates exhibited positive cytotoxicity. High cytotoxicity values correlated to the presence of stx genes. The majority of hly-positive but stx-negative environmental isolates also exhibited a certain degree of cytotoxicity. Isolates were also tested for sorbitol utilization and were genotyped by ribotyping and by repetitive extragenic palindromic PCR (REP-PCR) as potential means of quickly identifying virulent strains from the environment, but none of these methods could be used to distinguish cytotoxic environmental isolates. Only 31% of the isolates were negative for sorbitol fermentation, and none of the isolates had common ribotypes or REP-PCR fingerprints. This study suggests that overall higher cytotoxicity values correlated with the production of stx genes, and the majority of hly-positive but stx-negative environmental isolates also exhibited a certain degree of cytotoxicity. This study demonstrated that there is widespread distribution of potentially virulent E. coli strains in the environment that may be a cause of concern for human health.     

New Flagellin-Specifying Genes in Some Escherichia coli Strains

Data for further development of the flagellar antigen genetics of the species Escherichia coli are reported. Two new flagellin genes named fllA and flmA were found in E. coli 781-55, E2987-73, and E223-69, the test strains for E. coli flagellar antigens H44, H55, and H54, respectively (collection of the International Escherichia and Klebsiella Centre of the World Health Organization, Copenhagen, Denmark). Two alleles of fllA were identified that encode flagellar antigens H44 (fllA₄₄) and H55 (fllA₅₅), and the only flmA allele found (flmA₅₄) encodes antigen H54. The sites of their integration in the E. coli K-12 chromosome after P1-mediated transduction were approximately determined and found to be separate from each other and from the known regions of flagellar genes of E. coli and salmonellae. The region of flm₅₄ was found to repress the expression of some alleles of the flagellin gene fliC. In addition, cryptic genes encoding antigens H4 and H38 were found in phenotypically monophasic test strains 781-55 and E2987-73, respectively.     

Prevalence of the stx2 Gene in Coliform Populations from Aquatic Environments

Shiga toxin-producing Escherichia coli strains are human pathogens linked to hemorrhagic colitis and hemolytic uremic syndrome. The major virulence factors of these strains are Shiga toxins Stx1 and Stx2. The majority of the genes coding for these toxins are borne by bacteriophages. Free Stx2-encoding bacteriophages have been found in aquatic environments, but there is limited information about the lysogenic strains and bacteria present in the environment that are susceptible to phage infection. The aim of this work was to study the prevalence and the distribution of the stx₂ gene in coliform bacteria in sewage samples of different origins. The presence of the stx₂ gene was monitored every 2 weeks over a 1-year period in a municipal sewage treatment plant. A mean value of 10² genes/ml was observed without significant variation during the study period. This concentration was of the same order of magnitude in raw municipal sewage of various origins and in animal wastewater from several slaughterhouses. A total of 138 strains carrying the stx₂ gene were isolated by colony hybridization. This procedure detected approximately 1 gene-carrying colony per 1,000 fecal coliform colonies in municipal sewage and around 1 gene-carrying colony per 100 fecal coliform colonies in animal wastewaters. Most of the isolates belonged to E. coli serotypes other than E. coli O157, suggesting a low prevalence of strains of this serotype carrying the stx₂ gene in the wastewater studied.     

Engineering of a robust Escherichia coli chassis and exploitation for large-scale production processes

In large-scale bioprocesses microbes are exposed to heterogeneous substrate availability reducing the overall process performance. A series of deletion strains was constructed from E. coli MG1655 aiming for a robust phenotype in heterogeneous fermentations with transient starvation. Deletion targets were hand-picked based on a list of genes derived from previous large-scale simulation runs. Each gene deletion was conducted on the premise of strict neutrality towards growth parameters in glucose minimal medium. The final strain of the series, named E. coli RM214, was cultivated continuously in an STR-PFR (stirred tank reactor – plug flow reactor) scale-down reactor. The scale-down reactor system simulated repeated passages through a glucose starvation zone. When exposed to nutrient gradients, E. coli RM214 had a significantly lower maintenance coefficient than E. coli MG1655 (Δm_s = 0.038 g_Glucose/g_CDW/h, p < 0.05). In an exemplary protein production scenario E. coli RM214 remained significantly more productive than E. coli MG1655 reaching 44% higher eGFP yield after 28 h of STR-PFR cultivation. This study developed E. coli RM214 as a robust chassis strain and demonstrated the feasibility of engineering microbial hosts for large-scale applications.     

Immunogenicity of Salmonella enterica serovar Enteritidis virulence protein,InvH, and cross-reactivity of its antisera with Salmonella strains

Acellular vaccines containing bacterial immunodominant components such as surface proteins may be potent alternatives to live attenuated vaccines in order to reduce salmonellosis risk to human health. invH gene, an important part of needle complex in type three secretion system (TTSS) plays important role in efficient bacterial adherence and entry into epithelial cells. In this work we hypothesize that use of a 15 kDa recombinant InvH as Salmonella enterica serovar Enteritidis surface protein could provoke antibody production in mouse and would help us study feasibility of its potential for diagnosis and/or a recombinant vaccine. The purified InvH provoked significant rise of IgG in mice. Active protection induced by immunization with InvH against variable doses of S. enterica serovar Enteritidis, indicated that the immunized mice were completely protected against challenge with 10⁴ LD₅₀. The immunoreaction of sera from immunized mice with other Salmonella strains or cross reaction with sera of Salmonella strains inoculated mice is indicative of possessing by Salmonella strains of the surface protein, InvH, that can be employed in both prophylactic and diagnostic measures against S. enterica. Bacteria free spleen and ileum of the immunized mice in this study indicate that the invH gene affects bacterial invasion. Efficacy of the virulence protein, InvH, in shuttling into host cells in injectisome of S. enterica serovar Enteritidis and inhibition of this phenomenon by active immunization was shown in this study. In conclusion immunization with InvH protein can develop protection against S. enterica serovar Enteritidis infections. InvH in Salmonella strains can be exploited in protective measures as well as a diagnostic tool in Salmonella infections.     

The Early Innate Response of Chickens to Salmonella enterica Is Dependent on the Presence of O-Antigen but Not on Serovar Classification

Salmonella vaccines used in poultry in the EU are based on attenuated strains of either Salmonella serovar Enteritidis or Typhimurium which results in a decrease in S. Enteritidis and S. Typhimurium but may allow other Salmonella serovars to fill an empty ecological niche. In this study we were therefore interested in the early interactions of chicken immune system with S. Infantis compared to S. Enteritidis and S. Typhimurium, and a role of O-antigen in these interactions. To reach this aim, we orally infected newly hatched chickens with 7 wild type strains of Salmonella serovars Enteritidis, Typhimurium and Infantis as well as with their rfaL mutants and characterized the early Salmonella-chicken interactions. Inflammation was characterized in the cecum 4 days post-infection by measuring expression of 43 different genes. All wild type strains stimulated a greater inflammatory response than any of the rfaL mutants. However, there were large differences in chicken responses to different wild type strains not reflecting their serovar classification. The initial interaction between newly-hatched chickens and Salmonella was found to be dependent on the presence of O-antigen but not on its structure, i.e. not on serovar classification. In addition, we observed that the expression of calbindin or aquaporin 8 in the cecum did not change if inflammatory gene expression remained within a 10 fold fluctuation, indicating the buffering capacity of the cecum, preserving normal gut functions even in the presence of minor inflammatory stimuli.     

Predicting Essential Metabolic Genome Content of Niche-Specific Enterobacterial Human Pathogens during Simulation of Host Environments

Microorganisms have evolved to occupy certain environmental niches, and the metabolic genes essential for growth in these locations are retained in the genomes. Many microorganisms inhabit niches located in the human body, sometimes causing disease, and may retain genes essential for growth in locations such as the bloodstream and urinary tract, or growth during intracellular invasion of the hosts’ macrophage cells. Strains of Escherichia coli (E. coli) and Salmonella spp. are thought to have evolved over 100 million years from a common ancestor, and now cause disease in specific niches within humans. Here we have used a genome scale metabolic model representing the pangenome of E. coli which contains all metabolic reactions encoded by genes from 16 E. coli genomes, and have simulated environmental conditions found in the human bloodstream, urinary tract, and macrophage to determine essential metabolic genes needed for growth in each location. We compared the predicted essential genes for three E. coli strains and one Salmonella strain that cause disease in each host environment, and determined that essential gene retention could be accurately predicted using this approach. This project demonstrated that simulating human body environments such as the bloodstream can successfully lead to accurate computational predictions of essential/important genes.